Ion exchange chromatography using DEAE Cephadex

[daigoro]

My demonstrator left me on my own device to carry out the experiment. I've done this only once before and there wasn't a good protocol. Iam really unsure about what i am doing.
Here is the steps that I've done, if you can point out to me where i am wrong or I've missed something I want to thank you a million times:
-packing column with PB pH = 8
-load sample (lectin) in PBS =7,2 which has been dialysed
-elute in 5ml stepwise using PB =6
-measure OD of each fraction
-finish when OD is low enough.
Or you can give me a link to a detailed protocol. Thanks

[daigoro]

so it seems that I did not make myself very clear.
I am puzzled by the procedure I am following because the elution part does not look like those described in text book. Is this elution method stepwise or continuous?

[tom jupille]

Are you collecting 5 mL fractions? If so, the separation is "isocratic" (continuous). It sounds like the only reason for adding the eluant in 5mL "steps" is to make fraction collection easier.

However, some of your terms are new to me (I'm not a biochemist):
- what is "PB"?
- what is "PBS"?
- when you say "PB =6", did you mean "pH = 6"?

[daigoro]

PB = Phosphate buffer(mixture of two from Na2HPO4, NaH2PO4, K2HPO4 and KH2PO4)
PBS = Phosphate buffer saline (same as above with addition of saline)

[tom jupille]

Okay, are you collecting 5 mL fractions?